The present invention relates to a probe chip for assaying various detection items about objects to be detected, such as DNA, RNA and proteins, at a time (i.e., a many item-detecting sensor), and in particular to a polynucleotide probe chip for assaying DNA and a polynucleotide detection method using the same.
The genome project rapidly approaches the stage of functional genomics. DNA analysis has been clarifying the mechanism of livings and living phenomena. And the DNA analysis has been used for diagnosis of the many diseases. It is effective for understanding living phenomena and examining the effect of genes that the situation of expression of the genes is examined. As a high-powered means, a DNA probe array or a DNA chip wherein a great deal of DNA probes are divided depending on each kind thereof and fixed on the surface of a solid starts to be used. As a method for preparing this DNA chip, there are known a method of using photochemical reaction and lithography, which is widely used in the semiconductor industry, to synthesize oligomers having a designed sequence, one base by one base, on many cells divided up to areas (prior art 1: Science 251, pp. 767-773 (1991)); a method of planting a DNA probe one by one in every area (prior art 2: Proc. Natl. Acad. Sci. USA 93, pp. 4913-4918 (1996)); and the like. In order to increase the amount of probes fixed on a chip, a method is contrived wherein an acrylamide gel film is formed on a chip and probes are fixed on this gel (prior art 2). As a method for fixing DNA probes used in a DNA probe chip, there are known a method of using the bonding of biotin and avidin or fixing DNA probes on the surface of gold (Au) through a SH residue (prior art 3: Biophysical Journal 71, pp. 1079-1086 (1996)); a method of fixing DNA probes on the surface of glass (prior art 4: Analytical Biochemistry 247, pp. 96-101 (1997)); a method of fixing DNA probes on an element matrix of acrylamide gel applied on the surface of glass (prior art 2), and the like.
Not only DNA or its derivative but also RNA or its derivative may be held on the surface of a chip. Therefore, a chip which holds any polynucleotide on its surface is referred to as a polynucleotide probe chip, hereinafter. In both of the prior arts 1 and 2, there remains problems that their methods for producing DNA chips require much labor and time and costs for the production are high, in particular a problem that the production of a DNA chip composed of minutes parts having closely formed probe arrays requires far more labor and time. That is, usual users cannot form the chip easily. In examination using a DNA chip, there generally arises a serious problem that kinetics of hybridization is slow since a target DNA in a solution needs to be associated with probes held on the surface of a narrow chip by bringing a large amount of the solution into contact with the probes. In the case that the target DNA is long for the probes, it is necessary that the DNA approaches the held probes along such a sequence direction that the DNA becomes complementary to the probes. Therefore, kinetics of hybridization is especially slow. Since the area of the surface of the chip is limited, the amount of the held probes is such an amount as represented by fmol and the amount is restrictive. Thus, there remains a problem that when a large amount of DNAs having highly similar sequences are present, the detection of a target DNA of a very small amount is frequently disturbed. In the case that the amount of probes is small, falsely positive hybridization, i.e., a phenomenon that the probes on the surface of a chip are occupied by DNA having highly similar sequences arise easily. Among the above-mentioned problems, the assay using the DNA chip in the prior arts 1 and 2 has the problem that much time is required and high sensitivity is not attained. The prior art 2 has the problem that the diffusion of a sample DNA into gel in a chip results in a rate-determining step of hybridization, and also has the problem that it is a skilled job and difficult for usual users to make a polynucleotide probe chip holding probes uniformly and having reproducibility by treating gels formed on the surface of the chips chemically in such a manner that the probes can bond to the gels. In order to detect simultaneously fluorescence from respective areas in chips in the prior art, it is necessary to apply a laser beam widened by an expander from the same face at the side of a camera. High output is also necessary since the density of the laser beam applied to the respective areas drops. The laser beam which is reflected on the chip surface and then directly projected onto a detector results in a background. Thus, detection with high sensitivity is difficult. In the case of using chips in the prior art, therefore, it is general to use a method of scanning a laser beam by means of a laser scanning microscopy. As a result, there arises a problem that much time is required for assay. Furthermore, it is difficult to separate DNA captured in each of areas in chips in the prior art, dependently on each of the areas, and collect the DNA, dependently on each of sizes of DNA.
In order to overcome the above-mentioned problems, an object of the present invention is to provide a method for producing a polynucleotide probe chip which makes it possible to form desired polynucleotide probes in a close state easily and is low in production costs. Another object of the present invention is to provide a low-priced polynucleotide probe chip which causes an improvement in the kinetics of hybridization on the surface of the chip, makes high-sensitivity assay for a short time possible, and makes falsely positive hybridization less; and a method for detecting polynucleotide(s) and a polynucleotide detecting device which make it possible to separate DNA captured in each of areas in a chip, dependently on each of the areas, and collect the DNA, dependently on each of sizes of the DNA.
The polynucleotide probe chip of the present invention wherein plural areas holding different polynucleotide probes are arranged has the following features.
(A) Gels hold the polynucleotide probes in the respective areas. A polynucleotide sample is migrated in the gels in the areas by electrophoresis to hybridize the polynucleotide probes of the gels with sample polynucleotides. Thus, the possibility that the polynucleotide probe chips collide with the sample polynucleotides are raised, so that the kinetics of hybridization is made higher and the amount of the probes held by the gels can be increased.
(B) In actual use, sample polynucleotides labelled with a fluorophore are added to the polynucleotide probe chip wherein the areas holding the different polynucleotide probes are arranged, and then the polynucleotide sample is migrated in the gels of the respective gels by electrophoresis to hybridize the polynucleotide probes in the gels with specific polynucleotides. The specific polynucleotides captured in the respective areas can be detected by detecting the fluorophore- labelled polynucleotides captured in the gels of the respective areas of the polynucleotide probe chip.
The sample polynucleotides are not labelled in advance. First, the sample polynucleotide samples are added to the polynucleotide probe chip, and then they are migrated in the gels in the areas by electrophoresis, to hybridize the polynucleotide probes in the gels with specific polynucleotides. Next, DNTP labelled with a fluorophore by extension reaction using DNA polymerase or ddNTP labelled with a fluorophore is introduced to the polynucleotide probes hybridized with the polynucleotides in the areas of the gels in the polynucleotide probe chip, so that the polynucleotides are labelled. In this way, the specific polynucleotides captured in the respective areas can be detected.
In order to detect a DNA (cDNA) fragment having an unknown base sequence as a sample, for example, in mRNA-expression-profile assay, there is used a polynucleotide probe chip holding polynucleotide probes comprising a combination of a portion of a substantially common base sequence having from a 10-base sequence to a 60-base sequence, and any 2-base sequence or 3-base sequence for recognizing the fragment at the 3xe2x80x2 terminal thereof. Sample polynucleotides are added to the polynucleotide probe chip, and then they are migrated in the gels of the areas by electrophoresis, to hybridize the polynucleotide probes with the sample polynucleotides. Thus, hybrids are produced. The hybrids are detected by using DNA polymerase and introducing a fluorophore-labelled dNTP or a fluorophore-labelled ddNTP into the extended chains of the polynucleotide probes. Only the sample polynucleotide hybridized with the recognizing 2-base sequence or 3-base sequence at the 3xe2x80x2 terminal of the polynucleotide probes is detected by the existence of the fluorescence label. Alternatively, the complementary chain of the sample polynucleotide hybridized with the polynucleotide probe is synthesized by using the polynucleotide probe as a primer. The chain is modified to obtain an extended complementary chain composed of one chain. Next, this chain is hybridized with a fluorophore-labelled probe complementary to the extended complementary chain, and then the existence and the kind of hybridized sample are detected.
(C) A structure wherein electrodes are arranged in the chip makes it possible to migrate the sample polynucleotides in the gels, holding the polynucleotide probes, in the respective areas to hybridize the polynucleotide probes in the gels with the sample polynucleotides. In order to raise the kinetics of the hybridization to improve the efficiency of capturing specific polynucleotides, the sample polynucleotides are migrated in the same gels plural times to improve the efficiency of the hybridization.
(D) A polynucleotide probe chip wherein elements for detecting light are arranged or formed in respective areas of the surface of a substrate of the chip is used, and respective gels in the areas are irradiated along the direction parallel to the substrate surface with laser light. In this way, fluorescence from the gels in the areas can be simultaneously detected. This polynucleotide probe chip has the substrate on which a fine photodiode array is formed. Metallic vaporized layers through which light having a specific wavelength range transmits (fluorescence emitted from a fluorescence label) are formed on the respective photodiodes, and transparent electrodes are formed on the metallic vaporized layers through insulating layers. DNA probes are fixed onto the surfaces of the transparent electrodes. Wiring for detecting electric currents flowing through the respective photodiodes is formed on the surface of the polynucleotide probe chip. A reflective plate having a reflective face is pushed onto the upper surface of the polynucleotide probe chip in the manner that laser light is totally reflected. In the state that the reflective plate is pushed onto the gel surface in the respective areas, laser light is radiated thereto. Therefore, the laser light advances with total reflection being repeated, and is radiated along the direction substantially parallel to the substrate surface to the gels in the respective areas.
(E) A method for producing the polynucleotide probe chip is as follows. A monomer having a reaction residue and a probe set comprising plural kinds of polynucleotide probes having a residue bonded to the reaction residue of the monomer, and a chip comprising plural areas are beforehand prepared. Each kind of any probes selected from the polynucleotide probe set is mixed with the monomer, and the mixture is added to each of the different areas and gelatinized. By such a preparation, polynucleotide probes of a custom design can be held. As the monomer, acrylamide and derivatives thereof can be used. As the polynucleotide probes having a residue bonded to the reaction residue of the monomer, polynucleotide probes having an active vinyl residue such as an acryl residue can be used.
Of course, the polynucleotide probe held in each of the area is not limited to one kind. According to the purpose of analysis, a combination of plural kinds of the probes is held in each of the areas. A monomer having a reaction residue and a probe set comprising plural kinds of polynucleotide probes having a residue bonded to the reaction residue of the monomer, and a chip comprising plural areas are beforehand: prepared. Each kind of any probes selected from the polynucleotide probe set is mixed with the monomer, and the mixture is added to each of the different areas and gelatinized. By such a preparation, polynucleotide probes of a custom design can be prepared. If such a producing method is used, a constant volume of the probes is beforehand handled in the state of liquid and is held in the gel. Therefore, a polynucleotide probe chip which is uniform and is high in reproducibility can be produced. The method that the polynucleotide probes of the present invention are held by the polynucleotide probe chip is entirely different from that of the prior arts 2, 3 and 4.
According to the present invention, a polynucleotide probe of kind necessary for use can easily be arranged in the polynucleotide probe chip. The respective polynucleotide probes having a residue bonded to the reaction residue of the monomer can be prepared only by reacting polynucleotide having an amino residue and prepared in a DNA synthesizing device, N-acryloxysuccinimide, allylglycidyl ether and acrolein, and collecting the reactant product by gel filtration or ethanol precipitation. Thus, the probes are low-priced, and the polynucleotide probe chip itself can also be produced at a low price. The respective polynucleotide probes can be preserved.
According to the present invention, a probe array holding any kind of probe can easily be produced at a low-priced. Moreover, the DNA probes held in the gels and electrophoresis are used to migrate the sample in the gels forcibly. Accordingly, the probes held in the gels can be hybridized quickly and effectively with the sample DNA fragments. Since the DNA fragments which are not hybridized can be removed by electrophoresis, a background can be made lower by the present invention than by chips using gels in the prior art. In the present invention, the gels in all of the small holes (or each row of the small holes), or the gels on the surface of respective areas constituting a row can be simultaneously irradiated, from the side face of the chip, with light. Therefore, the time for scanning is unnecessary. Laser light is radiated in the direction that fluorescence is detected and in the direction perpendicular thereto. This is an arrangement that scatting light is not directly projected into a detector. The light emitted from the respective small holes can be simultaneously detected with a two-dimensional camera. Thus, assay with high-speed and high-sensitivity can be made. Furthermore, the respective holes (or the respective areas) can be irradiated simultaneously and effectively even by means of a sub-milliwatt class semiconductor laser.
An aspect of the present invention can be summarized as follows. Prepared are a monomer having a reaction residue,and a polynucleotide probe set comprising plural kinds of polynucleotide probes having a residue bonded to the reaction residue. The monomer is mixed with each kind of polynucleotide probes comprising any plural probes selected from the polynucleotide probe set. Each kind of the resultant mixtures is added to each of different small holes to gelatinize the mixture. Thus, a polynucleotide probe chip is produced. Sample DNA is forcibly migrated in the gels by electrophoresis. Laser light is projected onto the side face of the chip. The fluorescence emitted from the whole surface of the chip is collectively detected with a high-sensitive two-dimensional detector. Thus, it is possible to provide the polynucleotide probe chip for detecting DNA and the assay method, which have high hybridization-efficiency and make high-sensitivity and high-speed DNA detection possible.